The overall goal of this program is to develop a detailed molecular understanding of a novel post- translational targeting pathway that directs insertion of tail-anchored (TA) membrane proteins into the endoplasmic reticulum (ER) membrane. TA proteins constitute a large family of integral membrane proteins that play critical roles in virtually all aspects of cell biology, ranging from intracellular trafficking and viral replication to regulation of cell death. Despite the physiological importance of these proteins, the machinery and molecular mechanisms that mediate recognition, targeting, and insertion of TA proteins into the correct organellar membrane are poorly understood. The targeting and insertion of newly synthesized proteins into the ER membrane is an essential cellular process. For most membrane proteins, this is achieved co-translationally in a process mediated by the signal recognition particle. However, for the nearly 5% of all eukaryotic membrane proteins that are tail-anchored, targeting is achieved post-translationally. This process is mediated by a newly discovered and evolutionarily conserved ATPase, termed TRC40, which interacts with an ER-bound receptor to ensure efficient and accurate TA protein targeting. In an important breakthrough, we have recently determined high-resolution crystal structures of TRC40. Based upon this structural information, the following specific hypotheses will be tested. 1) Recognition of specific physiochemical properties of the targeting signal directs selective TA protein targeting by TRC40. 2) Conformational changes in TRC40 regulate its ATPase activity and TA substrate interactions. 3) The TRC receptor catalyzes these conformational changes through specific interactions with the TRC40-TA substrate complex. Using a powerful interdisciplinary approach, we will establish the fundamental biochemical and biophysical principles that underlie the process of TA protein targeting and insertion. The specific aims of this project are: 1. To determine the molecular basis of selective TA substrate recognition by TRC40 2. To determine how conformational changes in TRC40 regulate ATPase activity and TA substrate interactions 3. To establish how the TRC receptor coordinates targeting and release of TA proteins at the ER membrane PUBLIC HEALTH RELEVANCE: Tail-anchored (TA) membrane proteins play critical roles in virtually all aspects of cell biology. Given the role of TA proteins in pathologies ranging from cancer, neurodegenerative disease and chronic liver diseases (to name only a few), it is clear that a detailed mechanistic understanding of the targeting, membrane insertion and regulation of TA proteins is of fundamental cell biological and physiological significance. The studies described here will provide insight into the molecular details of TA membrane protein biogenesis, and may ultimately lead to the development of new therapeutic strategies that work by modulating the targeting of TA membrane proteins.